Biofuels in Perspective

204 Biofuels
NADH is that the reaction might be driven by reversed electron transport. This would require
that the NAD-dependent hydrogenase resides in the membrane. Such energy-driven
uphill oxidation of NADH has been described for Clostridium tetanomorphum, which contains
an NADH-dependent oxidoreductase that uses a sodium gradient to accomplish the
endergonic reduction of ferredoxin. Reduced ferredoxin is subsequently used to produce
H2. 36 However, an NADH-dependent hydrogenase has been purified from Thermoanaerobacter
tengcongensis, but this Fe-only hydrogenase is not membrane-bound. 28 Moreover,
Pyrococcus furiosus contains two NADPH-dependent hydrogenases, which are also not
membrane-bound. 37 The latter data suggest that hydrogen formation from NADH is not
necessarily dependent on an energy input via reversed electron transport, or at least not in
extreme- or hyperthermophiles.
Figure 11.2 illustrates that hydrogen formation from ferredoxin is thermodynamically
much more favorable, especially at elevated temperatures. At 70 ◦ C hydrogen formation
from ferredoxin is even exergonic in the presence of 1 atm of hydrogen.
11.4 Enzymology
Molecular hydrogen is produced by hydrogenases (EC 1.12.99.6, EC 1.12.7.2), which
catalyze the reduction of protons and thereby release the reducing equivalents that are
formed during the anaerobic degradation of organic substrates. Hydrogenases can also
perform the reverse reaction, which allows microorganisms to use H2 as a source of
reductant. Two main types of hydrogenases can be distinguished based on their metal
content, viz. the Fe-only hydrogenases and the [NiFe] hydrogenases. For details on the
structural composition and the catalytic mechanism of the various hydrogenases we refer
to some excellent reviews on these topics.
38– 41
Fe-only hydrogenases seem to be restricted to strictly anaerobes, whereas [NiFe] hydrogenases
are found more wide-spread in anaerobes, facultative anaerobes, and aerobes. Both
types of hydrogenase play a key role in the fermentative production of H2 which is discussed
here. As described above different electron carriers can deliver the electrons to the
terminal hydrogenase, viz. ferredoxin, NAD(H) or NADP(H), and these electron carriers
are reduced in a limited number of oxidation steps in the central metabolic pathways. The
two main oxidation steps during anaerobic sugar degradation (either EM- or PP-pathway)
are the conversion of glyceraldehyde-3-P to 3-P-glycerate and the conversion of pyruvate
to acetyl-CoA. Recycling of the reduced carriers occurs by different enzymatic steps.
Generally, reduced ferredoxin is used for proton reduction. As shown above, this is an
energetically favorable reaction which can be catalyzed by cytoplasmic monomeric Feonly
hydrogenases (e.g. Cl. pasteurianum) 42 or by multisubunit membrane-bound [NiFe]
hydrogenases. 41 Some of these [NiFe] hydrogenases are able to convert energy and their
six-subunit basic structure shows resemblance to the catalytic core of the NADH:quinone
oxidoreductase (Complex I). Several of the hydrogen-producing microorganisms discussed
here harbor such an energy-conserving [NiFe] hydrogenase (Table 11.3).
Hydrogen formation from NADH requires an NADH-dependent hydrogenase, which
has recently been characterized from Ta. tengcongensis (for details see also paragraph on
Thermoanaerobacter). 28 Homologs of the gene encoding this Fe-only hydrogenase can
be identified in Thermoanaerobacter ethanolicus, Caldicellulosiruptor saccharolyticus,